BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma

Abstract

The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.

Keywords: Brd2; Brd4; JQ1; mouse models; vorinostat.

Fig. 1.

Features of RVX2135, a novel BET inhibitor. (A) Structure of the chemical scaffold from which RVX2135 was developed. (B) FRET assay dose–response curves showing displacement of BET proteins from an acetylated histone-derived peptide by RVX2135. Data are mean values ± SD (error bars). (C) λ663 cells were treated for 24 h with the indicated concentrations of BET inhibitors JQ1 and RVX2135. Total protein lysates, the soluble fraction, and the insoluble fraction of detergent-generated lysates were loaded on a gel for Western blot analysis using the indicated antibodies. The method is described in greater detail in Fig. S1.

Fig. 2.

BET inhibitors induce cell-cycle arrest and apoptosis of Myc-transgenic mouse lymphoma cells. (A) λ820 cells were treated for 48 h with different concentrations of BETi and counted by trypan blue exclusion. Low concentrations of BETi were defined as 100 nM JQ1 and 1 µM RVX2135, whereas high concentrations were defined as 1 µM JQ1 and 10 µM RVX2135. Errors bars represent the standard deviation from three independent experiments. *P < 0.05. (B) λ820 cells were treated for 24 and 48 h with the indicated concentrations of BETi in the presence or absence of the pan-caspase inhibitor Q-VD-OPH. Cell-cycle distribution was analyzed by flow cytometry of 7-AAD–stained cells. Numbers show the proportion of cells in S phase. Quantifications of S phase and apoptosis (sub-G1) in three independent experiments are presented in Fig. S1 A and B. (C) λ820 cells were treated for 24 h with the indicated concentrations of BETi. Cells were lysed and analyzed by Western blotting for cleaved caspase 3 and cleaved PARP, markers of apoptosis. β-Actin was used as a loading control.

Fig. 3.

RVX2135 causes potent therapeutic responses in mouse models of aggressive Myc-induced lymphoma. (A) λ820 cells were transplanted into syngenic B6 mice via tail vein injection. Four days after injection, mice were dosed with 75 mg/kg [twice a day (b.i.d.); 5 d/wk) RVX2135 (n = 6) or vehicle (n = 7). Mice were monitored daily for signs of lymphoma (visible palpable lymphomas, panting suggesting thymic lymphoma, or overall health appearance), and four of the vehicle-treated mice and all of RVX2135-treated mice were killed when they showed signs of disease. The three remaining vehicle-treated mice were used in an experiment shown in Fig. S5C. (B) A lymphoma arising in a λ-Myc mouse (ID 2749) was transplanted into recipient B6 mice via tail vein injection accompanied by treatment with either vehicle or RVX2135. Four days after injection, mice were dosed with 75 mg/kg b.i.d. RVX2135 (n = 8) or vehicle (n = 9). Mice were monitored daily for signs of lymphoma and were killed when they showed signs of disease. (C) Four mice were transplanted with lymphoma cells from a λ-Myc mouse (2749). Twelve days after transplantation, when mice were yet to show manifest disease, they were injected with [¹⁸F]FDG and scanned with a PET/computed tomography imager. All mice had a strong signal in the spleen and in several lymph nodes. The signal ratio comparing normal tissue (skeletal muscle) and lymphoma was calculated. Mice were treated with four doses of 75 mg/kg b.i.d. RVX2135 and imaged posttherapy. The tumor PET signal-to-background ratio (TBR) of all measured PET-positive sites (n = 23), representing the spleens of all mice and four or five lymph nodes, is shown. All point (Fig. 3C) are individual data point. **P < 0.01. (D) Representative PET image of one mouse before and after treatment with RVX2135. PET-positive lymphoid tissues used in the TBR calculation in C are shown with green (lymph nodes) and blue (spleen arrows).

Fig. 4.

RVX2135 causes apoptosis and rapid therapeutic responses in mouse models of aggressive Myc-induced lymphoma. (A and B) Mice transplanted with λ820 cells (A) or serially transplanted with lymphoma from mouse 2749 (B) were monitored for elevated leukocyte count (white blood cells; WBCs) in the blood drawn from a hind leg vein (vena saphena). When counts had reached significantly above normal mouse blood WBCs (5–15 cells per nL), the mice were randomized to treatment with either vehicle or 75 mg/kg RVX2135 (b.i.d.). Mice in groups of three were killed 2 h after one to five bidaily doses; shown here are the experiments where significant reductions in spleen size had occurred (four doses during 36 h for λ820 cells and five doses during 48 h for 2749). After sacrifice, spleens were weighed and subsequently fixed in formalin and processed for TUNEL staining or immunohistochemistry to detect cleaved caspase 3, markers of apoptosis. Counts are either from whole sections (caspase 3; n = 6) or representative fields of view (TUNEL; n = 6). Representative images are shown in Fig. S5. (C) λ-Myc mice were interbred to Cdkn2a knockout mice to generate λ-Myc;Cdkn2a^+/− mice. These were followed for signs of lymphoma. Typically at 40–50 d of age the mice developed lymphoma with an associated leukocytosis, which is an acceleration of a similar rate to that seen in Eµ-Myc;Cdkn2a^+/− mice and λ-Myc;p53^+/− mice (–52). (D) Seven λ-Myc;Cdkn2a^+/− mice exhibiting leukocytosis (>20 WBCs per nL), as assessed by WBC count of peripheral blood, were treated with 75 mg/kg b.i.d. RVX2135 for 3 d. Six hours after the last dose, blood samples were drawn and a WBC count was performed. Six out of seven mice responded, and data were analyzed by a paired t test. Errors bars represent the standard deviation. *P < 0.05, **P < 0.01.

Fig. 5.

BETi induce broad transcriptional effects affecting several growth-promoting gene signatures. (A) Supervised hierarchical clustering of Illumina BeadChip microarray data in λ820 and Eµ239 cells treated with BETi. Shown are the 50 most down- and up-regulated genes (fold change). Red color indicates induced expression, black means unchanged expression, and green indicates down-regulated expression. Some of the genes up-regulated by BET inhibitor treatment were confirmed by qRT-PCR (Fig. S7). (B) Venn diagram of the genes down-regulated by treatment with λ820 and Eµ239 cells with 1 µM JQ1 for 24 h. Data of average expression levels (n = 2) in Eµ239 cells are present in Dataset S2. (C) Gene set enrichment analysis of genes coregulated (down) by JQ1 in both λ820 and Eµ239 cells. Shown are top transcription factors (TFs) associated with the gene signatures. GSEA of genes shared and uniquely regulated in λ820 and Eµ239 cell lines is shown in Dataset S3. FDR, false discovery rate; q-value, the statistical significance of an FDR test. (D) Venn diagram showing the similarities and differences in the expression profiles of λ820 cells treated with the indicated concentrations of BETi or HDACi for 24 h. Data of average expression levels are presented in Datasets S1 and S4.

Fig. 6.

BETi and HDACi induce transcription of stress-regulated genes. (A) Quantitative RT-PCR analyses of Egr1, Bbc3 (Puma), and Cd74 expression in λ820 cells treated with the indicated compounds. (B and C) ChIP assay experiment of cells treated with the indicated compounds. Following ChIP, qPCR analyses were performed using primers directed against the promoter region of Cd74 (B) or the indicated regions of Egr1 (green) or Bbc3 (red). Shown are the mean values from three independent experiments of relative binding compared in DMSO-treated cells. Errors bars represent the standard deviation from three independent experiments. (D) Venn diagram showing the similarities between genes induced in multiple myeloma MM1.S cells treated with 500 nM JQ1 for 6 h (GEO dataset GSE44931) and λ820 cells treated with 1 µM JQ1 or 1 µM vorinostat for 24 h. (E) IGV screenshot showing gene occupancy of the indicated proteins in ChIP-seq data of the TXNIP gene.

Fig. 7.

Effects of BETi are similar to effects of HDAC inhibitors. (A) λ820 cells were treated for 24 and 48 h with the indicated concentrations of HDACi vorinostat or LBH-589. Cell-cycle distribution was analyzed by flow cytometry of 7-AAD–stained cells. Numbers show the proportion of cells in S phase. Additional HDACi used are shown in Fig. S11A. (B) λ820 cells were treated for 24 h with the indicated concentrations of HDACi and counted by using the gate of the viable cells in the flow cytometer. (C) λ820 cells were treated for 24 h with 1 µM JQ1 or 10 µM RVX2135 monotherapy or in combination with 1 µM HDAC inhibitor vorinostat. Cell-cycle distribution was analyzed by flow cytometry of 7-AAD–stained cells. Numbers show the proportion of cells in S phase. Data of S phase and apoptosis (sub-G1) from three independent experiments are shown in Fig. S11B. (D) Mice carrying 2749 lymphoma cells were acutely treated with bidaily oral doses of 75 mg/kg RVX2135, daily i.p. injections of 40 mg/kg vorinostat, or a combination of both treatments when showing WBC counts suggestive of leukocytosis. Single-treated mice were treated until WBC counts started to increase again and/or palpable lymphomas appeared. Combination-treated mice were maintained on treatment for an additional week, at which time they were scored as disease-free by palpation and WBC count. They were then taken off treatment until they showed signs of lymphoma and were killed. Shown in the graphs is the average ± SD, five mice per treatment group. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8.

BET inhibitors cause major effects on transcription of growth-promoting genes. (A) Summary of effects of BETi in cancer cells. Depending on which genes get suppressed and/or activated, the cells either undergo cell-cycle arrest and apoptosis (Myc-induced lymphoma) or only undergo cell-cycle arrest (e.g., melanoma). (B) BETi cause major repression of transcription by blocking Brd4–p-TEFb–mediated pause release. Furthermore, some genes are induced, likely because of repositioning of p-TEFb and RNA pol II to stress-induced genes such as p53 target genes, immediate early genes (Egr1, Fos, Jun), histone genes, and HEXIM1, a negative regulator of p-TEFb. (C) Schematic model, based on data presented herein (Fig. 6), publicly available ChIP-seq data (Fig. S9), and conclusions from published studies in the cancer and HIV fields. (Left) In untreated cells, Brd4–p-TEFb mediate the phosphorylation of Ser2 on the RNA pol II C-terminal domain. This results in elongation of paused transcription. Upon BET inhibitor treatment, Brd4 can no longer bind acetylated histones, resulting in transcriptional pausing of many genes, including those needed for cell proliferation. HDAC inhibitor treatment does not result in suppression of the same genes, as judged by microarray. (Center) Stress-induced genes are stimulated by both BETi and HDACi via Brd4-dependent (e.g., Egr1 and TXNIP) and Brd4-independent (Bbc3 and JUN) mechanisms. The Brd4-independent mechanism may involve a transient release of p-TEFb from HEXIM1–7S RNP complexes, as seen in induction of HIV transcription by BETi and HDACi and another BET protein. (Right) Genes that are not pause-regulated in a BET-dependent manner can continue transcription or even enhance it (Fig. S9, histone genes). Whether or not this represents an active event or simply reflects enhanced availability of p-TEFb and RNA pol II as suggested in B remains to be elucidated.